Identification of a muscle-specific isoform of VMA21 as a potent actor in X-linked myopathy with excessive autophagy pathogenesis.

Defective lysosomal acidification is responsible for a large range of multi-systemic disorders associated with impaired autophagy. Diseases caused by mutations in the VMA21 gene stand as exceptions, specifically affecting skeletal muscle (X-linked Myopathy with Excessive Autophagy, XMEA) or liver (Congenital Disorder of Glycosylation). VMA21 chaperones vacuolar (v-) ATPase assembly, which is ubiquitously required for proper lysosomal acidification. The reason VMA21 deficiencies affect specific, but divergent tissues remains unknown. Here, we show that VMA21 encodes a yet-unreported long protein isoform, in addition to the previously described short isoform, which we name VMA21-120 and VMA21-101, respectively. In contrast to the ubiquitous pattern of VMA21-101, VMA21-120 was predominantly expressed in skeletal muscle, and rapidly up-regulated upon differentiation of mouse and human muscle precursors. Accordingly, VMA21-120 accumulated during development, regeneration and denervation of mouse skeletal muscle. In contrast, neither induction nor blockade of autophagy, in vitro and in vivo, strongly affected VMA21 isoform expression. Interestingly, VMA21-101 and VMA21-120 both localized to the sarcoplasmic reticulum of muscle cells, and interacted with the v-ATPase. While VMA21 deficiency impairs autophagy, VMA21-101 or VMA21-120 overexpression had limited impact on autophagic flux in muscle cells. Importantly, XMEA-associated mutations lead to both VMA21-101 deficiency and loss of VMA21-120 expression. These results provide important insights into the clinical diversity of VMA21-related diseases and uncover a muscle-specific VMA21 isoform that potently contributes to XMEA pathogenesis.

Association between ZASP/LDB3 Pro26Ser and Inclusion Body Myopathy.

Inclusion body myositis (IBM) is a slowly progressive disorder belonging to the idiopathic inflammatory myopathies, and it represents the most common adult-onset acquired myopathy. The main clinical features include proximal or distal muscular asymmetric weakness, with major involvement of long finger flexors and knee extensors. The main histological findings are the presence of fiber infiltrations, rimmed vacuoles, and amyloid inclusions. The etiopathogenesis is a challenge because both environmental and genetic factors are implicated in muscle degeneration and a distinction has been made previously between sporadic and hereditary forms. Here, we describe an Italian patient affected with a hereditary form of IBM with onset in his mid-forties. Next-generation sequencing analysis disclosed a heterozygous mutation c.76C>T (p.Pro26Ser) in the PDZ motif of the LDB3/ZASP gene, a mutation already described in a family with a late-onset myopathy and highly heterogenous degree of skeletal muscle weakness. In the proband's muscle biopsy, the expression of ZASP, myotilin, and desmin were increased. In our family, in addition to the earlier age of onset, the clinical picture is even more peculiar given the evidence, in one of the affected family members, of complete ophthalmoplegia in the vertical gaze. These findings help extend our knowledge of the clinical and genetic background associated with inclusion body myopathic disorders.

Protein Aggregates and Aggrephagy in Myopathies.

A number of muscular disorders are hallmarked by the aggregation of misfolded proteins within muscle fibers. A specialized form of macroautophagy, termed aggrephagy, is designated to remove and degrade protein aggregates. This review aims to summarize what has been studied so far about the direct involvement of aggrephagy and the activation of the key players, among others, p62, NBR1, Alfy, Tollip, Optineurin, TAX1BP1 and CCT2 in muscular diseases. In the first part of the review, we describe the aggrephagy pathway with the involved proteins; then, we illustrate the muscular disorder histologically characterized by protein aggregates, highlighting the role of aggrephagy pathway abnormalities in these muscular disorders.

p.Asn1180Ile mutation of SCN4A gene in an Italian family with myopathy and myotonic syndrome.

INTRODUCTION: Mutations of the skeletal muscle sodium channel gene SCN4A are associated with several neuromuscular disorders including hyper/hypokaliemic periodic paralysis, paramyotonia congenita and sodium channel myotonia. These disorders are distinguished from dystrophic myotonias by the absence of progressive weakness and extramuscular systemic involvement. METHODS: We present an Italian family with 2 subjects carrying a p.Asn1180Ile mutation in SCN4A gene showing a peculiar clinical picture characterized by the association of myopathic features and myotonia. RESULTS: The clinical, electromyographic and histological findings of these patients are reported. The possible pathogenicity of the mutation was tested by three different software, all giving positive results. DISCUSSION: This is the first report of a dominant, heterozygous mutation in SCN4A causing a complex phenotype of non-congenital myopathy and myotonic syndrome. We suggest that, in patients with myotonia and myopathy not related to dystrophic myotonias, the sequence analysis of SCN4A gene should be performed.

Opposing roles of miR-21 and miR-29 in the progression of fibrosis in Duchenne muscular dystrophy.

Excessive extracellular matrix deposition progressively replacing muscle fibres is the endpoint of most severe muscle diseases. Recent data indicate major involvement of microRNAs in regulating pro- and anti-fibrotic genes. To investigate the roles of miR-21 and miR-29 in muscle fibrosis in Duchenne muscle dystrophy, we evaluated their expression in muscle biopsies from 14 patients, and in muscle-derived fibroblasts and myoblasts. In Duchenne muscle biopsies, miR-21 expression was significantly increased, and correlated directly with COL1A1 and COL6A1 transcript levels. MiR-21 expression was also significantly increased in Duchenne fibroblasts, more so after TGF-ß1 treatment. In Duchenne fibroblasts the expression of miR-21 target transcripts PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SPRY-1 (Sprouty homolog 1) was significantly reduced; while collagen I and VI transcript levels and soluble collagen production were significantly increased. MiR-29a and miR-29c were significantly reduced in Duchenne muscle and myoblasts, and miR-29 target transcripts, COL3A1, FBN1 and YY1, significantly increased. MiR-21 silencing in mdx mice reduced fibrosis in the diaphragm muscle and in both Duchenne fibroblasts and mdx mice restored PTEN and SPRY-1 expression, and significantly reduced collagen I and VI expression; while miR-29 mimicking in Duchenne myoblasts significantly decreased miR-29 target transcripts. These findings indicate that miR-21 and miR-29 play opposing roles in Duchenne muscle fibrosis and suggest that pharmacological modulation of their expression has therapeutic potential for reducing fibrosis in this condition.

A rare mutation in MYH7 gene occurs with overlapping phenotype.

Mutations in the beta-myosin heavy chain gene (MYH7) cause different muscle disorders. The specific molecular pathobiological processes that cause these different phenotypes remains unexplained. We describe three members of a family with an autosomal dominant mutation in the distal rod of MYH7 [c.5401G> A (p.Glu1801Lys)] displaying a complex phenotype characterized by Laing Distal Myopathy like phenotype, left ventricular non compaction cardiomyopathy and Fiber Type Disproportion picture at muscle biopsy. We suggest that this overlapping presentation confirm the phenotypic variability of MYH7 myopathy and may be helpful to improve the genotype phenotype correlation.

Fibrosis and inflammation are greater in muscles of beta-sarcoglycan-null mouse than mdx mouse.

The Sgcb-null mouse, with knocked-down ß-sarcoglycan, develops severe muscular dystrophy as in type 2E human limb girdle muscular dystrophy. The mdx mouse, lacking dystrophin, is the most used model for Duchenne muscular dystrophy (DMD). Unlike DMD, the mdx mouse has mild clinical features and shows little fibrosis in limb muscles. To characterize ECM protein deposition and the progression of muscle fibrosis, we evaluated protein and transcript levels of collagens I, III and VI, decorin, and TGF-ß1, in quadriceps and diaphragm, at 2, 4, 8, 12, 26, and 52 weeks in Sgcb-null mice, and protein levels at 12, 26, and 52 weeks in mdx mice. In Sgcb-null mice, severe morphological disruption was present from 4 weeks in both quadriceps and diaphragm, and included conspicuous deposition of extracellular matrix components. Histopathological features of Sgcb-null mouse muscles were similar to those of age-matched mdx muscles at all ages examined, but, in the Sgcb-null mouse, the extent of connective tissue deposition was generally greater than mdx. Furthermore, in the Sgcb-null mouse, the amount of all three collagen isoforms increased steadily, while, in the mdx, they remained stable. We also found that, at 12 weeks, macrophages were significantly more numerous in mildly inflamed areas of Sgcb-null quadriceps compared to mdx quadriceps (but not in highly inflamed regions), while, in the diaphragm, macrophages did not differ significantly between the two models, in either region. Osteopontin mRNA was also significantly greater at 12 weeks in laser-dissected highly inflamed areas of the Sgcb-null quadriceps compared to the mdx quadriceps. TGF-ß1 was present in areas of degeneration-regeneration, but levels were highly variable and in general did not differ significantly between the two models and controls. The roles of the various subtypes of macrophages in muscle repair and fibrosis in the two models require further study. The Sgcb-null mouse, which develops early fibrosis in limb muscles, appears more promising than the mdx mouse for probing pathogenetic mechanisms of muscle fibrosis and for developing anti-fibrotic treatments. Highlights • The Sgcb-null mouse develops severe muscular dystrophy, the mdx mouse does not. • Fibrosis developed earlier in Sgcb-null quadriceps and diaphragm than mdx. • Macrophages were commoner in mildly inflamed parts of Sgcb-null quadriceps than mdx. • The Sgcb-null model appears more useful than mdx for studying fibrotic mechanisms. • The Sgcb-null model also appears more useful for developing anti-fibrotic treatments.

Duchenne muscular dystrophy fibroblast nodules: a cell-based assay for screening anti-fibrotic agents.

Severe muscle fibrosis is the endpoint of many chronic myopathies. Identification of factors that regulate fibrosis is important for understanding its pathogenesis and for developing anti-fibrotic treatments that prevent muscle destruction. We have developed an in vitro model for screening potential anti-fibrotic agents. The model consists of three-dimensional clusters (nodules) of fibroblasts derived from Duchenne muscular dystrophy (DMD) muscle. The primary fibroblasts spontaneously and quickly form nodules resembling fibrotic foci (cells plus extracellular matrix) when grown on a solid substrate. We tested the anti-fibrotic action of suramin, decorin, and spironolactone (all with established anti-fibrotic activity) on the model. All three agents significantly reduced nodule number, and spironolactone and suramin significantly reduced nodule diameter. Nodule secretion of soluble collagen was also significantly reduced by decorin and spironolactone treatment, whereas suramin had no significant effect. Collagen I and fibronectin protein expression was significantly reduced in the culture medium of control and DMD fibroblasts by spironolactone treatment, but not by decorin and suramin treatment. Finally, in DMD fibroblast monolayers, collagen deposition was significantly reduced by all three agents. Spironolactone significantly reduced collagen I and fibronectin transcript levels, whereas decorin reduced only fibronectin. Our in vitro model of fibrogenesis has thus revealed differing anti-fibrotic effects in the three anti-fibrotic agents tested. It therefore appears as a useful and sensitive system for the testing of anti-fibrotic drugs and could be adapted for the high-throughput screening of new anti-fibrotic molecules.

Novel DPAGT1 Gene Mutation in Two Twins with Congenital Myasthenic Syndrome and a Review of the Literature.

Congenital myasthenic syndromes (CMS) are rare diseases caused by mutation in genes coding for proteins involved in neuromuscular junction structure and function. DPAGT1 gene mutations are a rare cause of CMS whose clinical evolution and pathophysiological mechanisms have not been clarified completely. We present the case of two twins displaying an infancy-onset predominant limb-girdle phenotype and carrying a novel DPAGT1 mutation associated with unusual histological and clinical findings. CMS can mimic paediatric and adult limb-girdle phenotype, hence neurophysiology plays a fundamental role in the differential diagnosis.

VCP-related myopathy: a case series and a review of literature.

The valosin-containing protein (VCP), a widely expressed protein, controls the ubiquitin-proteasome system, endolysosomal sorting, and autophagy to maintain cellular proteostasis. Frontotemporal dementia (FTD), inclusion body myopathy, and Paget's disease of the bone (PDB) are all caused by dominant missense mutations in the VCP gene, which interfere with these mechanisms and cause a multisystem proteinopathy. We describe phenotypic and genetic findings of five patients with four different mutations in VCP gene (NM_007126): c.278G > A (p.R93H), c.463C > T (p.R155C), c.410C > T (p.P137L), c.464G > A (p.R155H), c.410C > T (p.P137L). We analysed the patient' biopsies, all characterized by a muscular phenotype, and we executed immunofluorescence staining to evaluate the presence of proteins: p62, VCP, desmin, myotilin, TDP-43. Eventually we performed a brief literature review to compare our cases with those already reported. Our report strongly suggest that VCP gene mutations can be related with a predominant skeletal muscle phenotype without any central nervous system involvement, as occasionally reported in the literature. Particularly, our patient with R93H shows only myopathic involvement while this mutation has been described once associated only to Hereditary Spastic Paraplegia. Further study will be necessary to understand such a broad and different clinical spectrum.

Kv1.1 knock-in ataxic mice exhibit spontaneous myokymic activity exacerbated by fatigue, ischemia and low temperature.

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by myokymia and attacks of ataxic gait often precipitated by stress. Several genetic mutations have been identified in the Shaker-like K(+) channel Kv1.1 (KCNA1) of EA1 individuals, including V408A, which result in remarkable channel dysfunction. By inserting the heterozygous V408A, mutation in one Kv1.1 allele, a mouse model of EA1 has been generated (Kv1.1(V408A/+)). Here, we investigated the neuromuscular transmission of Kv1.1(V408A/+) ataxic mice and their susceptibility to physiologically relevant stressors. By using in vivo preparations of lateral gastrocnemius (LG) nerve-muscle from Kv1.1(+/+) and Kv1.1(V408A/+) mice, we show that the mutant animals exhibit spontaneous myokymic discharges consisting of repeated singlets, duplets or multiplets, despite motor nerve axotomy. Two-photon laser scanning microscopy from the motor nerve, ex vivo, revealed spontaneous Ca(2+) signals that occurred abnormally only in preparations dissected from Kv1.1(V408A/+) mice. Spontaneous bursting activity, as well as that evoked by sciatic nerve stimulation, was exacerbated by muscle fatigue, ischemia and low temperatures. These stressors also increased the amplitude of compound muscle action potential. Such abnormal neuromuscular transmission did not alter fiber type composition, neuromuscular junction and vascularization of LG muscle, analyzed by light and electron microscopy. Taken together these findings provide direct evidence that identifies the motor nerve as an important generator of myokymic activity, that dysfunction of Kv1.1 channels alters Ca(2+) homeostasis in motor axons, and also strongly suggest that muscle fatigue contributes more than PNS fatigue to exacerbate the myokymia/neuromyotonia phenotype. More broadly, this study points out that juxtaparanodal K(+) channels composed of Kv1.1 subunits exert an important role in dampening the excitability of motor nerve axons during fatigue or ischemic insult.

Osteopontin is highly expressed in severely dystrophic muscle and seems to play a role in muscle regeneration and fibrosis.

AIMS: To increase our understanding of profibrotic mechanisms in dystrophic muscle. METHODS AND RESULTS: Extracellular matrix, fibrosis-related molecules and histopathology were assessed in skeletal muscle of patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy type 1A (MDC1A).Osteopontin expression was much higher in DMD and MDC1A than in BMD and control muscle. Osteopontin was expressed in mononuclear cell infiltrates, on some muscle fibre surfaces, in regenerating fibres, and in calcified fibres. In all pathological muscles, matrix metalloproteinase (MMP)-1 was increased around groups of fibres that were also characterized by absence of collagen 1. The amounts of MMP-2, MMP-9 and tissue inhibitor of MMP -1 transcripts were also increased, whereas their proteins were variably expressed in muscle fibres (surface or cytoplasm) and at foci of necrosis and regeneration. Inflammatory cells, fibroblasts and myofibroblasts were more numerous in DMD and MDC1A than in BMD muscle. CONCLUSIONS: Several fibrosis-related factors are greatly altered in severely dystrophic skeletal muscle. Osteopontin was the most conspicuously upregulated, both as transcript and as protein, in muscle fibres and infiltrating cells, indicating an intimate involvement in fibrosis, and also in inflammation and muscle regeneration, although its precise roles in these processes remain to be elucidated.

Gain-of-Function STIM1 L96V Mutation Causes Myogenesis Alteration in Muscle Cells From a Patient Affected by Tubular Aggregate Myopathy.

Tubular Aggregate Myopathy (TAM) is a hereditary ultra-rare muscle disorder characterized by muscle weakness and cramps or myasthenic features. Biopsies from TAM patients show the presence of tubular aggregates originated from sarcoplasmic reticulum due to altered Ca2+ homeostasis. TAM is caused by gain-of-function mutations in STIM1 or ORAI1, proteins responsible for Store-Operated-Calcium-Entry (SOCE), a pivotal mechanism in Ca2+ signaling. So far there is no cure for TAM and the mechanisms through which STIM1 or ORAI1 gene mutation lead to muscle dysfunction remain to be clarified. It has been established that post-natal myogenesis critically relies on Ca2+ influx through SOCE. To explore how Ca2+ homeostasis dysregulation associated with TAM impacts on muscle differentiation cascade, we here performed a functional characterization of myoblasts and myotubes deriving from patients carrying STIM1 L96V mutation by using fura-2 cytofluorimetry, high content imaging and real-time PCR. We demonstrated a higher resting Ca2+ concentration and an increased SOCE in STIM1 mutant compared with control, together with a compensatory down-regulation of genes involved in Ca2+ handling (RyR1, Atp2a1, Trpc1). Differentiating STIM1 L96V myoblasts persisted in a mononuclear state and the fewer multinucleated myotubes had distinct morphology and geometry of mitochondrial network compared to controls, indicating a defect in the late differentiation phase. The alteration in myogenic pathway was confirmed by gene expression analysis regarding early (Myf5, Mef2D) and late (DMD, Tnnt3) differentiation markers together with mitochondrial markers (IDH3A, OGDH). We provided evidences of mechanisms responsible for a defective myogenesis associated to TAM mutant and validated a reliable cellular model usefull for TAM preclinical studies.

Altered production of extra-cellular matrix components by muscle-derived Duchenne muscular dystrophy fibroblasts before and after TGF-beta1 treatment.

To probe pro-fibrotic mechanisms in dystrophic muscle, we isolated primary fibroblasts from Duchenne muscular dystrophy (DMD) and control muscle biopsies and induced transdifferentiation in myofibroblasts by transforming growth factor beta1 (TGF-beta1) treatment. We compared proliferating activity, soluble collagen production, and transcript and protein levels of decorin, myostatin, TGF-beta1, matrix metalloproteinase-1 (MMP-1; interstitial collagenase), MMP-2 (gelatinase), MMP-3 (stromelysin), MMP-7 (matrilysin), and the tissue inhibitors of metalloproteinases inhibitors (TIMPs) 1-4, in fibroblasts and myofibroblasts. Principal differences included a significantly greater proliferation rate and soluble collagen production, a significant upregulation of decorin, myostatin and MMP-7 transcripts and proteins, and a significant downregulation of MMP-1 and TIMP-3 transcripts (with MMP-1 protein being reduced as shown by enzyme-linked immunosorbent assay and TIMP-3 protein apparently being reduced on Western blot), in untreated DMD fibroblasts compared with controls. TGF-beta1 transdifferentiation significantly lowered decorin and myostatin and significantly increased TGF-beta1 transcript and protein, significantly increased MMP-1 and TIMP-3, and significantly lowered MMP-7 transcript and protein in DMD cells compared with pretreatment controls. The differences between DMD and control fibroblasts showed that DMD fibroblasts had a profibrotic phenotype, accentuated by TGF-beta1 treatment. Dystrophin absence itself could exert a direct influence on the homeostasis of the extracellular matrix (ECM) by allowing leakage of cellular components to the extracellular space or by abnormal cellular uptake of extracellular growth factors, cytokines, or enzymes influencing muscle fibroblasts either directly by altering adhesion properties or indirectly by interactions with molecules released into the ECM by muscle or inflammatory cells. The transdifferentiation of muscle fibroblasts might serve as a simplified model of fibrosis for further elucidation of the mechanisms of muscle fibrosis and for testing possible anti-fibrotic agents.

Improved Criteria for the Classification of Titin Variants in Inherited Skeletal Myopathies.

BACKGROUND: Extensive genetic screening results in the identification of thousands of rare variants that are difficult to interpret. Because of its sheer size, rare variants in the titin gene (TTN) are detected frequently in any individual. Unambiguous interpretation of molecular findings is almost impossible in many patients with myopathies or cardiomyopathies. OBJECTIVE: To refine the current classification framework for TTN-associated skeletal muscle disorders and standardize the interpretation of TTN variants. METHODS: We used the guidelines issued by the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) to re-analyze TTN genetic findings from our patient cohort. RESULTS: We identified in the classification guidelines three rules that are not applicable to titin-related skeletal muscle disorders; six rules that require disease-/gene-specific adjustments and four rules requiring quantitative thresholds for a proper use. In three cases, the rule strength need to be modified. CONCLUSIONS: We suggest adjustments are made to the guidelines. We provide frequency thresholds to facilitate filtering of candidate causative variants and guidance for the use and interpretation of functional data and co-segregation evidence. We expect that the variant classification framework for TTN-related skeletal muscle disorders will be further improved along with a better understanding of these diseases.

The Genetic Landscape of Dystrophin Mutations in Italy: A Nationwide Study.

Dystrophinopathies are inherited diseases caused by mutations in the dystrophin (DMD) gene for which testing is mandatory for genetic diagnosis, reproductive choices and eligibility for personalized trials. We genotyped the DMD gene in our Italian cohort of 1902 patients (BMD n = 740, 39%; DMD n =1162, 61%) within a nationwide study involving 11 diagnostic centers in a 10-year window (2008-2017). In DMD patients, we found deletions in 57%, duplications in 11% and small mutations in 32%. In BMD, we found deletions in 78%, duplications in 9% and small mutations in 13%. In BMD, there are a higher number of deletions, and small mutations are more frequent than duplications. Among small mutations that are generally frequent in both phenotypes, 44% of DMD and 36% of BMD are nonsense, thus, eligible for stop codon read-through therapy; 63% of all out-of-frame deletions are eligible for single exon skipping. Patients were also assigned to Italian regions and showed interesting regional differences in mutation distribution. The full genetic characterization in this large, nationwide cohort has allowed us to draw several correlations between DMD/BMD genotype landscapes and mutation frequency, mutation types, mutation locations along the gene, exon/intron architecture, and relevant protein domain, with effects on population genetic characteristics and new personalized therapies.

Exome sequencing detects compound heterozygous nonsense LAMA2 mutations in two siblings with atypical phenotype and nearly normal brain MRI.

LAMA2 mutations cause the most frequent congenital muscular dystrophy subtype MDC1A and a variety of milder phenotypes, characterized by total or partial laminin-α2 deficiency. In both severe and milder cases brain MRI invariably shows abnormal white matter signal intensity. We report clinical, histopathological, imaging and genetic data on two siblings with very subtle, and at first undetected, reduction in laminin-α2 expression, and brain MRI showing minor non-specific abnormalities. Clinical features in the female proband were characterized by muscle weakness involving neck and axial muscles, and pelvic girdle and distal lower limb muscles, reduced tendon reflexes and pes cavus. Clinical features in a younger brother were similar, and remained stable in both siblings during the follow up. Whole exome sequencing (WES) detected two heterozygous truncating LAMA2 mutations. Brain MRI in combination with laminin-α2 immunohistochemistry might not be sufficient and WES might be the only means to reach a diagnosis.

A novel mutation in the N-terminal acting-binding domain of Filamin C protein causing a distal myofibrillar myopathy.

Variants in Filamin C (FLNC) gene may cause either cardiomyopathies or different myopathies. We describe a family affected by a distal myopathy with autosomal dominant inheritance. The onset of the disease was in the third decade with gait impairment due to distal leg weakness. Subsequently, the disease progressed with an involvement of proximal lower limbs and hand muscles. Muscle biopsy, performed in one subject,identified relevant myofibrillar abnormalities. We performed a target gene panel testing for myofibrillar myopathies by NGS approach which identified a novel mutation in exon 3 of FLNC gene (c.A664G:p.M222V), within the N-terminal actin-binding (ABD) domain. This variant has been identified in all affected members of the family, thus supporting its pathogenic role. Differently from previously identified variants, our family showed a predominant leg involvement and myofibrillar aggregates, thus further expanding the spectrum of Filamin C related myopathies.